Demystifying Multicolor Flow Cytometry

Flow cytometry has come a long way in the 80 years since its inception. The now highly sophisticated technique is an indispensable tool for the diagnosis, prognosis, and monitoring of patients with hematologic malignancies and a range of other disorders. Yet, its use began during the 1930s as a relatively simple, automated way to count red blood cells: A red blood cell suspension is passed through a capillary tube and each cell is accounted for by a photoelectric apparatus attached to a microscope.1

In 1949, engineer and inventor Wallace Coulter discovered a method for detecting and counting cells or particles in suspension as each passed through an aperture in an electric current, which eventually became known as the Coulter Principle. In 1953, he patented an apparatus that incorporated this principle, the Coulter Counter, that still is used today for characterizing human blood cells.2

German scientist Wolfgang Göhde, PhD, further refined the technique and added new detection capabilities such as measurement of size and granularity by light scatter and fluorescence. He is credited with developing the first commercial fluorescence-based flow cytometry device in 1968.3

Today’s state-of-the-art flow cytometers are multicolor flow cytometry instruments that go well beyond the ability to count cells, allowing clinicians and researchers to detect a combination of as many as 10 or 12 cell surface and cytoplasmic markers – all on the same cell from a patient’s blood sample.

“For the field of hematologic malignancies, multicolor flow cytometry is one of the best tools we have for subtyping malignancies,” says David L. Jaye, MD, a pathologist specializing in lymphoid malignancies and an assistant professor in the department of pathology and laboratory medicine at the Emory University School of Medicine in Atlanta.

ASH Clinical News spoke with Dr. Jaye and other researchers about the principles behind multicolor flow cytometry, its clinical applications in hematology and oncology, and the evolving role of this powerful diagnostic tool.

Flow Cytometry 101

Flow cytometry is performed on a laboratory machine called the fluorescence activated cell sorter, commonly called FACS, which counts cells and particles, measures their forward and side scatter properties, and detects fluorescence linked to these cells using lasers. The dyes, called fluorochromes or fluorophores, are fluorescent, chemical compounds that can re-emit light upon excitation.

Following a technique developed during the 1940s, these dyes are conjugated to monoclonal antibodiesbind to antigens on the surface of cells or in the cell’s cytoplasm.4 As its name implies, the FACS is used to identify and count the cells present in a patient sample, but it also can be used to physically sort cells for further analyses.

Only a single fluorochrome was used to label the antibodies initially, but today, there are tens of fluorochromes in use. Each emits light at different wavelengths, which allows researchers to incubate cell samples with multiple fluorochrome-antibody combinations – as many as 10 for clinical applications and even more for research applications – facilitating the detailed characterization of subpopulations of cells within a sample.

“We can look at multiple antigens on the same cell and the process is automated, removing the chance of human error.”

—David L. Jaye, MD

With the use of multiple fluorochromes, the process and analysis become more challenging. Ideally, there will be minimal spectral overlap among the wavelengths emitted by each fluorochrome, which avoids confounding the instrument’s ability to distinguish among cell populations. But there is some room for spectral overlap, because today’s machines can correct for this wavelength overlap, called “spillover.”

“We’ve been doing flow cytometry for hematopathology for decades and, 20 years ago, we were only able to label cells from a patient sample with a few different colors,” explained Tracy I. George, MD, professor of pathology at the University of Utah School of Medicine and a medical director of hematopathology at ARUP Laboratories in Salt Lake City, Utah. The limited number of colors meant that pathologists could answer only simple questions, like “whether the patient had leukemia or lymphoma cells present.”

Two decades later, “most laboratories are doing 10-color flow cytometry and labeling a single cell with up to eight antibodies, plus forward light scatter and side light scatter,” she continued. “Eight- and 10-color flow cytometry are reliable tools and are now considered the standard in the industry.”

To prepare a patient cell sample for multicolor flow cytometry, the sample is processed to make the cells permeable, allowing the added antibodies to both bind to antigens on the cell surface and within the cytoplasm – all on the same cell. At the same time, this allows scientists to assess the cell’s geometric properties. “We’re getting information on the size of the cell, as well as its internal complexity, so that you can tell a malignant lymphocyte from a neutrophil, for example,” said Dr. George.

Smaller Samples, More Information

“The power of flow cytometry is that it gives us information about the cell lineage – lymphoid or myeloid, B cell or T cell,” Dr. George commented. “We also can figure out the cells’ state of differentiation, whether these are immature cells, like leukemic blasts from the bone marrow, or more fully differentiated cells, like a mature B-cell lymphoma.” The antibodies used to detect certain cell markers also provide information for clinicians about the presence of either wild-type or abnormal or malignant cells.

Pathologists use the flow cytometry–generated data to assess whether the populations of cells from the patient contain malignant cells. Together with histopathology data, this information informs a diagnosis.

“Flow cytometry is ordered upfront to make a diagnosis,” Dr. Jaye said. Various institutions, including the World Health Organization, have specified criteria for the diagnosis of hematologic malignancies using distinct markers.5 For example, for acute myeloid leukemia, pathologists look for cytoplasmic myeloperoxidase, which points to a myeloid lineage. For T-cell lymphomas, they look for the CD3 surface antigen, and for B-cell malignancies, they look for surface CD19 and cytoplasmic CD22 and CD79A.

The other important technique for immunophenotyping a patient’s blood or bone marrow sample is immunohistochemistry, which also uses antibodies to detect specific antigens within a sample to identify and differentiate malignant and nonmalignant cells. Immunohistochemistry has the advantage of allowing pathologists to assess the architecture and structure of the tissue, according to Dr. Jaye.

But the important malignant cells might be few and far between and in the clinical setting, and immunohistochemistry allows labeling of one slide with only a single antibody. This means that pathologists need to make qualitative assessments to understand if a certain cell on one slide is the same type of cell that is labeled with a different antibody on another slide. “That can be frustrating,” Dr. Jaye noted, “but, with flow cytometry, we can look at multiple antigens on the same cell and the process is automated, removing the chance of human error.”

Mike Keeney, associate scientist at the Lawson Research Institute in London, Ontario, Canada, agreed. “Flow cytometry is a more definitive methodology. Pathologists looking under a microscope need to evaluate whether what they are looking at are malignant cells,” he said. “Flow cytometry gives us an objective assessment that is not based on an individual’s judgment.”

The advent of multicolor flow cytometry actually has decreased the amount of a patient sample needed for processing. “What’s so wonderful is that we don’t need more sample to assess more markers,” Dr. George said. “It’s quite the opposite: It’s fewer cells for more information.”

“We only need 1 or 2 milliliters of cerebrospinal fluid to determine whether a malignancy has invaded the central nervous system,” Mr. Keeney added. “This was not possible before.” Flow cytometry also is allowing pathologists to move away from having to remove a patient’s entire lymph node to test for malignant cells, to taking a small, fine aspirate sample for diagnosis and analysis, in some cases.

From Diagnosis to Prognosis

Aside from diagnosis, multicolor flow cytometry is used in the clinic for other aspects of the management of patients with hematologic malignancies, including prognosis and therapy selection. Often, the pathologist is making a diagnosis, assessing prognostic markers, and determining which could be used for therapy selection – all at the same time. For example, if a diagnostic flow cytometry analysis reveals that a patient has CD30-positive T-cell lymphoma, the presence of the CD30 marker also indicates that the patient is eligible for therapy with brentuximab vedotin, a CD30-targeting antibody drug conjugate.

The antibodies used to detect malignancy-specific and nonspecific markers can also be used for prognosis. Whether a patient with CD30-positive anaplastic large cell lymphoma also harbors a rearrangement in the anaplastic lymphoma kinase (ALK) gene can help clinicians with a prognosis, because patients with ALK-negative anaplastic large cell lymphoma have less favorable outcomes and poorer survival.6

“We are often making the diagnosis and looking at prognostic and therapeutic markers, as well,” Dr. George said. “We select the markers to analyze on the basis of whether the patient is suspected to have a lymphoma or leukemia, for example. Then, based on the initial results, we may add on additional markers for more detailed characterization.”

An Emerging Application: Minimal Residual Disease Detection

Detection and monitoring of minimal residual disease (MRD) is now becoming a standard practice for patients with hematologic malignancies, including for acute lymphocytic leukemia, acute myeloid leukemia, and multiple myeloma.7,8 MRD refers to the counting of malignant cells that remain after a patient has completed a course of therapy. Depending on the malignancy, MRD typically is assessed via samples from a blood draw or a bone marrow aspirate.

MRD detection allows clinicians to evaluate a therapy’s efficacy, can help select subsequent therapy options, and serves as a prognostic marker – providing an indicator of how long a patient’s remission might last, how deep the remission is, and when and if a patient is likely to relapse.

Flow cytometry is one of the sensitive methods used to detect MRD, along with DNA-sequencing technologies that detect the DNA unique to malignant cells. Using flow cytometry to measure MRD requires a larger sample than that needed for other research applications, since the technician is looking for the likely-rare malignant cell that could have remained following therapy, which requires scanning many more normal cells.

“There are now guidelines for detection of MRD in patients with certain hematologic malignancies,” said Dr. George, referring to the International Clinical Cytometry Society’s consensus recommendations.9 There are only a few labs in the U.S. that represent the “go-to” experts for MRD detection and serve as reference labs for other clinical centers. The efforts to standardize the technique have come from the flow cytometry community itself. “More and more, labs want to do MRD-testing, and it has become clear that, frankly, some labs weren’t doing this well. Now, we have rigorous criteria for how to set up MRD-testing at an institution. We are actually just in the process of finalizing this at our center,” he stated.

“So far, only a subset of extensively experienced pathologists specializes in MRD detection because it is so technically challenging,” Dr. George noted.

For Dr. Jaye, one advantage of using flow cytometry for MRD detection is that the cell detection technique, unlike DNA-sequencing methods, does not require the technician to know the patient’s original diagnosis. To use DNA sequencing, clinicians need to look for specific mutations present in the malignant cells. “But, with flow cytometry, we can identify malignant cells without knowing the patient’s diagnosis or the mutations found in their malignant cells, because we are simply looking for those cells that are not normal in the population,” he explained.

New Therapies, New Challenges

One challenge of using flow cytometry for MRD detection or standard disease-tracking is working around the effects of newer targeted treatments. For example, if a patient is treated with an anti-CD30 antibody, the CD30-expressing cells may no longer be present following treatment. The diseased cell population shifts in their marker expression profile, requiring new antibodies against new markers for the cells’ detection.

“When clinicians treat a multiple myeloma patient with an anti-CD38 therapy, for example, that results in loss and masking of that CD38 antigen,” Dr. Jaye explained. “So, when we analyze a post-therapy biopsy, we will no longer have CD38 in our toolbox to recognize the malignant cells.” Scientists and pathologists now are trying to find the right combination of novel markers for post-treatment flow cytometry analyses.

Another challenge for MRD detection – in which technicians look for the rare “needle-in-the-haystack” diseased cell– is sample quality. “Bone marrow samples are, by nature, contaminated with peripheral blood cells, but some samples will be ‘bloodier’ than others,” said Dr. George. These contaminated samples make the detection of rare malignant cells especially challenging. “I’ve been a pathologist in a number of centers, and the quality of the sample is the biggest issue with flow cytometry everywhere. If it’s garbage in, it’s garbage out.”

There is also a lack of skilled pathologists and flow cytometry technicians who can perform diagnosis and follow-up of patients with hematologic malignancies, according to Dr. George. “It’s a big problem in the lab industry,” she said. “There is a shortage of medical technologists.” —By Anna Azvolinsky


References

  1. Moldovan A. Photo-electric technique for the counting of microscopical cells. Science. 1934;80:188-9.
  2. Smithsonian National Museum of American History. Coulter Counter-Model A. Accessed October 31, 2018, from http://americanhistory.si.edu/collections/search/object/nmah_1200679.
  3. Purdue University Cytometry Laboratories. Wolfgang Gö Accessed October 31, 2018, from http://www.cyto.purdue.edu/cdroms/cyto10a/seminalcontributions/gohde.html.
  4. Coons AH, Creech HJ, Jones RN. Immunological properties of an antibody containing a fluorescence group. Exptl Biol Med. 1941;47:200-2.
  5. Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood. 2008;111:3941-67.
  6. Hapgood G, Savage KJ. The biology and management of systemic anaplastic large cell lymphoma. Blood. 2015;126:17-25.
  7. Kim C, Delaney K, McNamara M, et al. Cross-sectional physician survey on the use of minimal residual disease testing in the management of pediatric and adult patients with acute lymphoblastic leukemia. Hematology. 2018;21:1-9.
  8. Roshal M. Minimal residual disease detection by flow cytometry in multiple myeloma: why and how? Semin Hematol. 2018;55:4-12.
  9. Arroz M, Came N, Lin P, et al. Consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting. Cytometry B Clin Cytom. 2016;90:31-9.

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